Avian polynucleotide vaccine formula

ABSTRACT

The avian vaccine formula comprises at least three polynucleotide vaccine valencies each comprising a plasmid integrating, so as to express it in vivo in the host cells, a gene with one avian pathogen valency, these valencies being selected from the group consisting of Marek&#39;s disease virus, Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, infectious anaemia virus, the plasmids comprising, for each valency, one or more of the genes selected from the group consisting of gB and gD for the Marek&#39;s disease virus, HN and F for the Newcastle disease virus, VP2 for the infectious bursal disease virus, S, M and N for the infectious bronchitis virus, C+NS1 for the infectious anaemia virus.

[0001] The present invention relates to a vaccine formula allowing thevaccination of avian species, in particular chickens. It also relates toa corresponding method of vaccination.

[0002] Associations of vaccines against a number of viruses responsiblefor pathologies in chicken have already been proposed in the past.

[0003] The associations developed so far were prepared from inactivatedvaccines or live vaccines. Their use poses problems of compatibilitybetween valencies and of stability. It is indeed necessary to ensureboth the compatibility between the different vaccine valencies, whetherfrom the point of view of the different antigens used from the point ofview of the formulations themselves. The problem of the conservation ofsuch combined vaccines and also of their safety especially in thepresence of an adjuvant also exists. These vaccines are in general quiteexpensive.

[0004] Patent applications WO-A-90 11092, WO-A-92 19183, WO-A-94 21797and WO-A-95 20660 have made use of the recently developed technique ofpolynucleotide vaccines. It is known that these vaccines use a plasmidcapable of expressing, in the host cells, the antigen inserted into theplasmid. All the routes of administration have been proposed(intraperitoneal, intravenous, intramuscular, transcutaneous,intradermal, mucosal and the like). Various vaccination means can alsobe used, such as DNA deposited at the surface of gold particles andprojected so as to penetrate into the animal's skin (Tang et al.,Nature, 356, 152-154, 1992) and liquid jet injectors which make itpossible to transfect at the same time the skin, the muscle, the fattytissues and the mammary tissues (Furth et al., Analytical Biochemistry,205, 365-368, 1992).

[0005] The polynucleotide vaccines may also use both naked DNAs and DNAsformulated, for example, inside lipids or cationic liposomes.

[0006] The invention therefore proposes to provide a multivalent vaccineformula which makes it possible to ensure vaccination against a numberof pathogenic avian viruses.

[0007] Another objective of the invention is to provide such a vaccineformula combining different valencies while exhibiting all the criteriarequired for mutual compatibility and stability of the valencies.

[0008] Another objective of the invention is to provide such a vaccineformula which makes it possible to combine different valencies in thesame vehicle.

[0009] Another objective of the invention is to provide such a vaccinewhich is easy and inexpensive to use.

[0010] Yet another objective of the invention is to provide a method forvaccinating Gallinaceans which makes it possible to obtain protection,including multivalent protection, with a high level of efficiency and oflong duration, as well as good safety and an absence of residues.

[0011] The subject of the present invention is therefore an avianvaccine formula comprising at least three polynucleotide vaccinevalencies each comprising a plasmid integrating, so as to express it invivo in the host cells, a gene with one avian pathogen valency, thesevalencies being selected from the group consisting of Marek's diseasevirus (MDV), Newcastle's disease virus (NDV) , infectious bursal diseasevirus (IBDV), infectious bronchitis virus (IBV), infectious anaemiavirus (CAV), infectious laryngotracheitis virus (ILTV),encephalomyelitis virus (AEV or avian leukosis virus ALV), pneumovirosisvirus, and avian plague virus, the plasmids comprising, for eachvalency, one or more of the genes selected from the group consisting ofgB and gD for the Marek's disease virus, HN and F for the Newcastledisease virus, VP2 for the infectious bursal disease virus, S, M and Nfor the infectious bronchitis virus, C+NS1 for the infectious anaemiavirus, gB and gD for the infectious laryngotracheitis virus, env andgag/pro for the encephalomyelitis virus, F and G for the pneumovirosisvirus and HA, N and NP for the avian plague virus.

[0012] Valency in the present invention is understood to mean at leastone antigen providing protection against the virus for the pathogenconsidered, it being possible for the valency to contain, as subvalency,one or more natural or modified genes from one or more strains of thepathogen considered.

[0013] Pathogenic agent gene is understood to mean not only the completegene but also the various nucleotide sequences, including fragmentswhich retain the capacity to induce a protective response. The notion ofa gene covers the nucleotide sequences equivalent to those describedprecisely in the examples, that is to say the sequences which aredifferent but which encode the same protein. It also covers thenucleotide sequences of other strains of the pathogen considered, whichprovide cross-protection or a protection specific for a strain or for astrain group. It also covers the nucleotide sequences which have beenmodified in order to facilitate the in vivo expression by the hostanimal but encoding the same protein.

[0014] Preferably, the vaccine formula according to the inventioncomprises three valencies chosen from Marek, infectious bursal,infectious anaemia and Newcastle. The infectious bronchitis valency canalso preferably be added thereto.

[0015] On this basis of 3, 4 or 5 valencies, it will be possible to addone or more of the avian plague, laryngotracheitis, pneumovirosis andencephalomyelitis valencies.

[0016] As regards the Marek valency, two genes may be used encoding gBand gD, in different plasmids or in one and the same plasmid. The use ofthe gB gene alone is however preferred.

[0017] For the Newcastle valency, the two HN and F chains, integratedinto two different plasmids or into one and the same plasmid, arepreferably used.

[0018] For the infectious bronchitis valency, the use of the S gene ispreferred. Optionally, but less preferably, S and M can be associated ina single plasmid or in different plasmids.

[0019] For the infectious anaemia valency, the two C and NS1 genes arepreferably associated in the same plasmid.

[0020] For the infectious laryngotracheitis valency, the use of the gBgene alone is preferred. Optionally, but less preferably, the two gB andgD genes can be associated in different plasmids or in one and the sameplasmid.

[0021] For the pneumovirosis valency, the use of the two F and G genes,in a single plasmid or in different plasmids, is preferred For the avianplague valency, the use of the HA gene is preferred. Optionally, butless preferably, it is possible to use the associations HA and NP or HAand N in different plasmids or in one and the same plasmid. Preferably,the HA sequences from more than one influenza virus strain, inparticular from the different strains found in the field, are preferablyassociated in the same vaccine. On the other hand, NP providescross-protection and the sequence from a single virus strain willtherefore be satisfactory.

[0022] For the encephalomyelitis valency, the use of env is preferred.

[0023] The vaccine formula according to the invention can be presentedin a dose volume of between 0.1 and 1 ml and in particular between 0.3and 0.5 ml.

[0024] The dose will be generally between 10 ng and 1 mg, preferablybetween 100 ng and 800 μg and preferably between 0.1 μg and 50 μg perplasmid type.

[0025] Use will be preferably made of naked plasmids, simply placed inthe vaccination vehicle which will be in general physiological salineand the like. It is of course possible to use all the polynucleotidevaccine forms described in the prior art and in particular formulated inliposomes.

[0026] Each plasmid comprises a promoter capable of ensuring theexpression of the gene inserted, under its control, into the host cells.This will be in general a strong eukaryotic promoter and in particular acytomegalovirus early CMV-IE promoter of human or murine origin, oroptionally of another origin such as rats, pigs and guinea pigs.

[0027] More generally, the promoter may be either of viral origin or ofcellular origin. As viral promoter other than CMV-IE, there may bementioned the SV40 virus early or late promoter or the Rous sarcomavirus LTR promoter. It may also be a promoter from the virus from whichthe gene is derived, for example the gene's own promoter.

[0028] As cellular promoter, there may be mentioned the promoter of acytoskeleton gene, such as, for example, the desmin promoter (Bolmont etal., Journal of Submicroscopic Cytology and Pathology, 1990, 22,117-122; and Zhenlin et al., Gene, 1989, 78, 243-254), or alternativelythe actin promoter.

[0029] When several genes are present in the same plasmid, these may bepresented in the same transcription unit or in two different units.

[0030] The combination of the different vaccine valencies according tothe invention may be preferably achieved by mixing the polynucleotideplasmids expressing the antigen(s) of each valency, but it is alsopossible to envisage causing antigens of several valencies to beexpressed by the same plasmid.

[0031] The subject of the invention is also monovalent vaccine formulaecomprising one or more plasmids encoding one or more genes from one ofthe viruses above, the genes being those described above. Besides theirmonovalent character, these formulae may possess the characteristicsstated above as regards the choice of the genes, their combinations, thecomposition of the plasmids, the dose volumes, the doses and the like.

[0032] The monovalent vaccine formulae may also be used (i) for thepreparation of a polyvalent vaccine formula as described above, (ii)individually against the actual pathology, (iii) associated with avaccine of another type (live or inactivated whole, recombinant,subunit) against another pathology, or (iv) as booster for a vaccine asdescribed below.

[0033] The subject of the present invention is in fact also the use ofone or more plasmids according to the invention for the manufacture ofan avian vaccine intended to vaccinate animals first vaccinated by meansof a first conventional vaccine (monovalent or multivalent) of the typein the prior art, in particular selected from the group consisting of alive whole vaccine, an inactivated whole vaccine, a subunit vaccine, arecombinant vaccine, this first vaccine having (that is to saycontaining or capable of expressing) the antigen(s) encoded by theplasmids or antigen(s) providing cross-protection.

[0034] Remarkably, the polynucleotide vaccine has a potent boostereffect which results in an amplification of the immune response and theacquisition of a long-lasting immunity.

[0035] In general, the first-vaccination vaccines can be selected fromcommercial vaccines available from various veterinary vaccine producers.

[0036] The subject of the invention is also a vaccination kit groupingtogether a vaccine formula according to the invention and afirst-vaccination vaccine as described above. It also relates to avaccine formula according to the invention accompanied by a leafletindicating the use of this formula as a booster for a first vaccinationas described above.

[0037] The subject of the present invention is also a method of avianvaccination, comprising the administration of an effective vaccineformula as described above. This vaccination method comprises theadministration of one or more doses of the vaccine formula, it beingpossible for these doses to be administered in succession over a shortperiod of time and/or in succession at widely spaced intervals.

[0038] The vaccine formulae according to the invention can beadministered in the context of this method of vaccination, by thedifferent routes of administration proposed in the prior art forpolynucleotide vaccination and by means of known techniques ofadministration.

[0039] The intramuscular route, the in ovo route, the intraocular route,nebulization and drinking water will be targeted in particular.

[0040] The efficiency of presentation of the antigens to the immunesystem varies according to the tissues. In particular, the mucousmembranes of the respiratory tree serve as barrier to the entry ofpathogens and are associated with lymphoid tissues which support localimmunity. In addition, the administration of a vaccine by contact withthe mucous membranes, in particular the buccal mucous membrane, thepharyngeal mucous membrane and the mucous membrane of the bronchialregion, is certainly of interest for mass vaccination.

[0041] Consequently, the mucosal routes of administration form part of apreferred mode of administration for the invention, using in particularneubilization or spray or drinking water. It will be possible to applythe vaccine formulae and the vaccination methods according to theinvention in this context.

[0042] The subject of the invention is also the method of vaccinationconsisting in making a first vaccination as described above and abooster with a vaccine formula according to the invention.

[0043] In a preferred embodiment of the process according to theinvention, there is administered in a first instance, to the animal, aneffective dose of the vaccine of the conventional, especiallyinactivated, live, attenuated or recombinant, type, or alternatively asubunit vaccine so as to provide a first vaccination, and, after aperiod preferably of 2 to 6 weeks, the polyvalent or monovalent vaccineaccording to the invention is administered.

[0044] The invention also relates to the method of preparing the vaccineformulae, namely the preparation of the valencies and mixtures thereof,as evident from this description.

[0045] The invention will now be described in greater detail with theaid of the embodiments of the invention taken with reference to theaccompanying drawings. List of figures FIG. No. 1: Plasmid pVR1012 FIG.No. 2: Plasmid pAB045 FIG. No. 3: Plasmid pAB080 FIG. No. 4: Sequence ofthe NBV HN gene, Texas GB strain FIG. No. 5: Plasmid pAB046 FIG. No. 6:Sequence of the NDV F gene, Texas GB strain FIG. No. 7: Plasmid pAB047FIG. No. 8: Sequence of the IBDV VP2 gene, Faragher strain FIG. No. 9:Plasmid pAB048 FIG. No. 10: Sequence of the IBV S gene, Massachusetts 41strain FIG. No. 11: Plasmid pAB049 FIG. No. 12: Sequence of the IBV Mgene, Massachusetts 41 strain FIG. No. 13: Plasmid pAB050 FIG. No. 14:Sequence of the IBV N gene, Massachusetts 41 strain FIG. No. 15: PlasmidpAB051 FIG. No. 16: Plasmid pAB054 FIG. No. 17: Plasmid pAB055 FIG. No.18: Plasmid pAB076 FIG. No. 19: Plasmid pAB089 FIG. No. 20: PlasmidpAB086 FIG. No. 21: Plasmid pAB081 FIG. No. 22: Plasmid pAB082 FIG. No.23: Plasmid pAB077 FIG. No. 24: Plasmid pAB078 FIG. No. 25: PlasmidpAB08B FIG. No. 26: Plasmid pAB079 Sequence listing SEQ ID No. SEQ IDNo. 1: Oligonucleotide AB062 SEQ ID No. 2: Oligonucleotide AB063 SEQ IDNo. 3: Oligonucleotide AB148 SEQ ID No. 4: Oligonucleotide AB149 SEQ IDNo. 5: Oligonucleotide AB072 SEQ ID No. 6: Oligonucleotide AB073 SEQ IDNo. 7: Sequence of the NDV HN gene, Texas GB strain SEQ ID No. 8:Oligonucleotide AB091 SEQ ID No. 9: Oligonucleotide AE092 SEQ ID No. 10:Sequence of the NDV F gene, Texas GB strain SEQ ID No. 11:Oligonucleotide AB093 SEQ ID No. 12: Oligonucleotide AE094 SEQ ID No.13: Sequence of the IBDV VP2 “gene”, Faragher strain SEQ ID No. 14:Oligonucleotide AB095 SEQ ID No. 15: Oligonucleotide PE096 SEQ ID No.16: Sequence of the IBV S gene, Massachusetts 41 strain SEQ ID No. 17:Oligonucleotide AB097 SEQ ID No. 18: Oligonucleotide AB098 SEQ ID No.19: Sequence of the IBV M gene, Massachusetts 41 strain SEQ ID No. 20:Oligonucleotide AB099 SEQ ID No. 21: Oligonucleotide AB100 SEQ ID No.22: Sequence of the IBV N gene, Massachusetts 41 strain SEQ ID No. 23:Oligonucleotide CD064 SEQ ID No. 24: Oligonucleotide CD065 SEQ ID No.25: Oligonucleotide CD066 SEQ ID No. 26: Oligonucleotide AB105 SEQ IDNo. 27: Oligonucleotide AB140 SEQ ID No. 28: Oligonucleotide AB141 SEQID No. 29: Oligonucleotide AB164 SEQ ID No. 30: Oligonucleotide AB165SEQ ID No. 31: Oligonucleotide AB160 SEQ ID No. 32: OligonucleotideAB161 SEQ ID No. 33: Oligonucleotide AB150 SEQ ID No. 34:Oligonucleotide AB151 SEQ ID No. 35: Oligonucleotide AB152 SEQ ID No.36: Oligonucleotide AB153 SEQ ID No. 37: Oligonucleotide AB142 SEQ IDNo. 38: Oligonucleotide AB143 SEQ ID No. 39: Oligonucleotide AB144 SEQID No. 40: Oligonucleotide AB145 SEQ ID No. 41: Oligonucleotide AB156SEQ ID No. 42: Oligonucleotide AB158 SEQ ID No. 43: OligonucleotideAB146 SEQ ID No. 44: Oligonucleotide AB147

EXAMPLE 1 Culture of the Viruses

[0046] The viruses are cultured on the appropriate cellular system untila cytopathic effect is obtained. The cellular systems to be used foreach virus are well known to persons skilled in the art. Briefly, thecells sensitive to the virus used, which are cultured in Eagle's minimumessential medium (MEM medium) or another appropriate medium, areinoculated with the viral strain studied using a multiplicity ofinfection of 1. The infected cells are then incubated at 37° C. for thetime necessary for the appearance of a complete cytopathic effect (onaverage 36 hours).

EXAMPLE 2 Extraction of the Viral Genomic DNAs

[0047] After culturing, the supernatant and the lysed cells areharvested and the entire viral suspension is centrifuged at 1000 g for10 minutes at +4° C. so as to remove the cellular debris. The viralparticles are then harvested by ultracentrifugation at 400,000 g for 1hour at +4° C. The pellet is taken up in a minimum volume of buffer (10mM Tris, 1 mM EDTA). This concentrated viral suspension is treated withproteinase K (100 μg/ml final) in the presence of sodium dodecylsulphate (SDS) (0.5% final) for 2 hours at 37° C. The viral DNA is thenextracted with a phenol/chloroform mixture and then precipitated with 2volumes of absolute ethanol. After leaving overnight at −20° C., the DNAis centrifuged at 10,000 g for 15 minutes at +4° C. The DNA pellet isdried and then taken up in a minimum volume of sterile ultrapure water.It can then be digested with restriction enzymes.

EXAMPLE 3 Isolation of the Viral Genomic RNAs

[0048] The RNA viruses were purified according to techniques well knownto persons skilled in the art. The genomic viral RNA of each virus wasthen isolated using the “guanidium thiocyanate/phenolchloroform”extraction technique described by P. Chromczynski and N. Sacchi (Anal.Biochem., 1987. 162, 156-159).

EXAMPLE 4 Molecular Biology Techniques

[0049] All the constructions of plasmids were carried out using thestandard molecular biology techniques described by J. Sambrook et al.(Molecular Cloning: A Laboratoxy Manual, 2nd Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989). All the restrictionfragments used for the present invention were isolated using the“Geneclean” kit (BIO 101 Inc. La Jolla, Calif.).

EXAMPLE 5 RT-PCR Technique

[0050] Specific oligonucleotides (comprising restriction sites at their5′ ends to facilitate the cloning of the amplified fragments) weresynthesized such that they completely cover the coding regions of thegenes which are to be amplified (see specific examples). The reversetranscription (RT) reaction and the polymerase chain reaction (PCR) werecarried out according to standard techniques (Sambrook J. et al., 1989).Each RT-PCR reaction was performed with a pair of specific amplimers andtaking, as template, the viral genomic RNA extracted. The complementaryDNA amplified was extracted with phenol/chloroform/isoamyl alcohol(25:24:1) before being digested with restriction enzymes.

EXAMPLE 6 Plasmid pVR1012

[0051] The plasmid pVR1012 (FIG. No. 1) was obtained from Vical Inc.,San Diego, Calif., USA. Its construction has been described in J.Hartikka et al. (Human Gene Therapy, 1996, 7, 1205-1217).

EXAMPLE 7 Construction of the Plasmid pAB045 (MDV gB Gene)

[0052] A PCR reaction was carried out with the Marek's disease virus(MDV) (RB1B strain) (L. Ross et al., J. Gen. Virol., 1989, 70,1789-1804) genomic DNA, prepared according to the technique in Example2, and with the following oligonucleotides:

[0053] AB062 (37 mer) (SEQ ID No. 1)

[0054] 5′ AAAACTGCAGACTATGCACTATTTTAGGCGGAATTGC 3′

[0055] AB063 (35 mer) (SEQ ID No. 2)

[0056] 5′ GGAAGATCTTTACACAGCATCATCTTTCTGAGTCTG 3′

[0057] so as to isolate the gene encoding the gB glycoprotein from theMDV virus in the form of a PstI-BglII fragment. After purification, the2613 bp PCR product was digested with PstI and Bg1I in order to isolatea 2602 bp PstI-BglII fragment. This fragment was ligated with the vectorpVR1012 (Example 6), previously digested with PstI and BglII, to givethe plasmid pAB045 (7455 bp) (FIG. No. 2).

EXAMPLE 8 Construction of the Plasmid pAB080 (MDV gD Gene)

[0058] A PCR reaction was carried out with the Marek's disease virus(MDV) (RB1B strain) (L. Ross et al., J. Gen. Virol., 1989, 72, 949-954)genomic DNA, prepared according to the technique in Example 2, and withthe following oligonucleotides:

[0059] AB148 (29 mer) (SEQ ID No. 3)

[0060] 5′ AAACTGCAGATGAAAGTATTTTTTTTTAG 3′

[0061] AB149 (32 mer) (SEQ ID No. 4)

[0062] 5′ GGAAGATCTTTATAGGCGGGAATATGCCCGTC 3′

[0063] so as to isolate the gene encoding the gD glycoprotein from theMDV virus in the form of a PstI-BglII fragment. After purification, the1215 bp PCR product was digested with PstI and BglII in order to isolatea 1199 bp PstI-BglII fragment. This fragment was ligated with the vectorpVR1012 (Example 6), previously digested with PstI and BglII, to givethe plasmid pAB080 (6051 bp) (FIG. No. 3).

EXAMPLE 9 Construction of the Plasmid pAB046 (NDV RN Gene)

[0064] An RT-PCR reaction according to the technique of Example 5 wascarried out with the Newcastle disease virus (NDV) (Texas GB strain)genomic RNA, prepared according to the technique of Example 3, and withthe following oligonucleotides:

[0065] AB072 (39 mer) (SEQ ID No. 5)

[0066] 5′ AGAATGCGGCCGCGATGGGCTCCAGATCTTCTACCAG 3′

[0067] AB094 (34 mer) (SEQ ID No. 6)

[0068] 5′ CGCGGATCCTTAAATCCCATCATCCTTGAGAATC 3′

[0069] so as to isolate the gene encoding the HN glycoprotein from theNDV virus, Texas GB strain (FIG. No. 4 and SEQ ID No. 7) in the form ofan NotI-BamHI fragment. After purification, the 1741 bp RT-PCR productwas digested with NotI and BamHI in order to isolate a 1723 bpNotI-BamHI fragment. This fragment was ligated with the vector pVR1012(Example 6), previously digested with NotI and BamHI, to give theplasmid pAB046 (6616 bp) (FIG. No. 5).

EXAMPLE 10 Construction of the Plasmid pAB047 (NDV F Gene)

[0070] An RT-PCR reaction according to the technique of Example 5 wascarried out with the Newcastle disease virus (NDV) (Texas GB strain)genomic RNA, prepared according to the technique of Example 3, and withthe following oligonucleotides:

[0071] AB091 (37 mer) (SEQ ID No. 8)

[0072] 5′ AGAATGCGGCCGCGATGGGCTCCAGATCTTCTACCAG 3′

[0073] AB092 (34 mer) (SEQ ID No. 9)

[0074] 5′ TGCTCTAGATCATATTTTTGTAGTGGCTCTCATC 3′

[0075] so as to isolate the gene encoding the F glycoprotein from theNDV virus, Texas GB strain (FIG. No. 6 and SEQ ID No. 10) in the form ofan NotI-XbaI fragment. After purification, the 1684 bp RT-PCR productwas digested with NotI and XbaI in order to isolate a 1669 bp NotI-XbaIfragment. This fragment was ligated with the vector pVR1012 (Example 6),previously digested with NotI and XbaI, to give the plasmid pAB047 (6578bp) (FIG. No. 7).

EXAMPLE 11 Construction of the Plasmid pAB048 (IBDV VP2 Gene)

[0076] An RT-PCR reaction according to the technique of Example 5 wascarried out with the infectious bursal disease virus (IBDV) (Faragherstrain) genomic RNA, prepared according to the technique of Example 3,and with the following oligonucleotides:

[0077] AB093 (33 mer) (SEQ ID No. 11)

[0078] 5′ TCAGATATCGATGACAAACCTGCAAGATCAAAC 3′

[0079] AB094 (38 mer) (SEQ ID No. 12)

[0080] 5′ AGAATGCGGCCGCTTACCTCCTTATAGCCCGGATTATG 3′

[0081] so as to isolate the sequence encoding the VP2 protein from theIBDV virus, Faragher strain (FIG. No. 8 and SEQ ID No. 13) in the formof an EcoRV-NotI fragment. After purification, the 1384 bp RT-PCRproduct was digested with EcoRV and NotI in order to isolate a 1367 bpEcoRV-NotI fragment. This fragment was ligated with the vector pVR1012(Example 6), previously digested with EcoRV and NotI, to give theplasmid pAB048 (6278 bp) (FIG. No. 9).

EXAMPLE 12 Construction of the plasmid pAB049 (IBV S1 Gene)

[0082] An RT-PCR reaction according to the technique of Example 5 wascarried out with the chicken infectious bronchitis virus (IPV)(Massachusetts 41 strain) genomic RNA, prepared according to thetechnique of Example 3, and with the following oligonucleotides:

[0083] AB095 (32 mer) (SEQ ID No. 14)

[0084] 5′ ACGCGTCGACATGTTGGTAACACCTCTTTTAC 3′

[0085] AB096 (35 mer) (SEQ ID No. 15)

[0086] 5′ GGAAGATCTTCATTAACGTCTAAAACGACGTGTTC 3′

[0087] so as to isolate the sequence encoding the S1 subunit of the Sglycoprotein from the IBV virus, Massachusetts 41 strain (FIG. No. 10and SEQ ID No. 16) in the form of a SalI-BglII fragment. Afterpurification, the 1635 bp RT-PCR product was digested with SalI andBglII in order to isolate a 1622 bp SalI-BglII fragment. This fragmentwas ligated with the vector pVR1012 (Example 6), previously digestedwith SalLI and BglII, to give the plasmid pAB049 (6485 bp) (FIG. No.11).

EXAMPLE 13 Construction of the Plasmid pAB050 (IBV M Gene)

[0088] An RT-PCR reaction according to the technique of Example 5 wascarried out with the chicken infectious bronchitis virus (IBV)(Massachusetts 41 strain) genomic RNA, prepared according to thetechnique of Example 3, and with the following oligonucleotides:

[0089] AB097 (37 mer) (SEQ ID No. 17)

[0090] 5′ ATAAGAATGCGGCCGCATGTCCAACGAGACAAATTGTAC 3′

[0091] AB098 (38 mer) (SEQ ID No. 18)

[0092] 5′ ATAAGAATGCGGCCGCTTTAGGTGTAAAGACTACTCCC 3′

[0093] so as to isolate the gene encoding the M glycoprotein from theIBV virus, Massachusetts 41 strain (FIG. No. 12 and SEQ ID No. 19) inthe form of a NotI-NotI fragment. After purification, the 710 bp RT-PCRproduct was digested with NotI in order to isolate a 686 bp NotI-NotIfragment. This fragment was ligated with the vector pVR1012 (Example 6),previously digested with NotI, to give the plasmid pAB050 (5602 bp)which contains the IBV M gene in the correct orientation relative to thepromoter (FIG. No. 13).

FIG. 14 Construction of the Plasmid pAB051 (IBV N Gene)

[0094] An RT-PCR reaction according to the technique of Example 5 wascarried out with the chicken infectious bronchitis virus (IBV)(Massachusetts 41 strain) genomic RNA, prepared according to thetechnique of Example 3, and with the following oligonucleotides:

[0095] AB099 (34 mer) (SEQ ID No. 20)

[0096] 5′ AAAACTGCAGTCATGGCAAGCGGTAAGGCAACTG 3′

[0097] AB100 (33 mer) (SEQ ID No. 21)

[0098] 5′ CGCGGATCCTCAAAGTTCATTCTCTCCTAGGGC 3′

[0099] so as to isolate the gene encoding the N protein from the IBVvirus, Massachusetts 41 strain (FIG. No. 14 and SEQ ID No. 22) in theform of a PstI-BamHI fragment. After purification, the 1250 bp RT-PCRproduct was digested with PstI and BamHI in order to isolate a 1233 bpPstI-BamHI fragment. This fragment was ligated with the vector pVR1012(Example 6), previously digested with PstI and BamHI, to give theplasmid pAB051 (6092 bp) (FIG. No. 15).

EXAMPLE 15 Construction of the Plasmid pAB054 (VAC VP1 Gene)

[0100] A PCR reaction was carried out with the chicken anaemia virus(CAV) (Cuxhaven-1 strain) genomic DNA (B. Meehan et al., Arch. Virol.,1992, 124, 301-319), prepared according to the technique of Example 2,and with the following oligonucleotides:

[0101] CD064 (39 mer) (SEQ ID No. 23)

[0102] 5′ TTCTTGCGGCCGCCATGGCAAGACGAGCTCGCAGACCGA 3′

[0103] CD065 (38 mer) (SEQ ID No. 24)

[0104] 5′ TTCTTGCGGCCGCTCAGGGCTGCGTCCCCCAGTACATG 3′

[0105] so as to isolate the gene encoding the CAV VP1 capsid protein inthe form of an NotI-NotI fragment. After purification, the 1377 bp PCRproduct was digested with NotI in order to isolate a 1359 bp NotI-NotIfragment. This fragment was ligated with the vector pVR1012 (Example 6),previously digested with NotI, to give the plasmid pAB054 (6274 bp)which contains the CAV VP1 gene in the correct orientation relative tothe promoter (FIG. No. 16).

EXAMPLE 17 Construction of the Plasmid pABb 055 (CAV VP2 Gene)

[0106] A PCR reaction was carried out with the chicken anaemia virus(CAV) (Cuxhaven-1 strain) genomic DNA (B. Meehan et al., Arch. Virol.,1992, 124, 301-319), prepared according to the technique of Example 2,and with the following oligonucleotides:

[0107] CD066 (39 mer) (SEQ ID No. 25)

[0108] 5′ TTCTTGCGGCCGCCATGCACGGGAACGGCGGACAACCGG 3′

[0109] AB105 (32 mer) (SEQ ID No. 26)

[0110] 5′ CGCGGATCCTCACACTATACGTACCGGGGCGG 3′

[0111] so as to isolate the gene encoding the CAV virus VP2 protein inthe form of an NotI-BamHI fragment. After purification, the 674 bp PCRproduct was digested with NotI and BamHI in order to isolate a 659 bpNotI-BamHI fragment. This fragment was ligated with the vector pVR1012(Example 6), previously digested with NotI and BamHI, to give theplasmid pAB055 (5551 bp) (FIG. No. 17).

EXAMPLE 18 Construction of the Plasmid pAB076 (ILTV gB Gene)

[0112] A PCR reaction was carried out with the chicken infectiouslaryngotracheitis virus (ILTV) (SA-2 strain) genomic DNA (K. Kongsuwanet al., Virology, 1991, 184, 404-410), prepared according to thetechnique of Example 2, and with the following oligonucleotides:

[0113] AB140 (38 mer) (SEQ ID No. 27)

[0114] 5′ TTCTTGCGGCCGCATGTCTTGAAAATGCTGATC 3′

[0115] AB141 (36 mer) (SEQ ID No. 28)

[0116] 5′ TTCTTGCGGCCGCTTATTCGTCTTCGCTTTCTTCTG 3′

[0117] so as to isolate the gene encoding the ILTV virus gB glycoproteinin the form of an NotI-NotI fragment. After purification, the 2649 bpPCR product was digested with NotI in order to isolate a 2631 bpNotI-NotI fragment. This fragment was ligated with the vector pVR1012(Example 6), previously digested with NotI, to give the plasmid pAB076(7546 bp) which contains the ILTV gB gene in the correct orientationrelative to the promoter (FIG. No. 18).

EXAMPLE 20 Construction of the Plasmid pAB089 (ILTV gD Gene)

[0118] A PCR reaction was carried out with the chicken infectiouslaryngotracheitis virus (ILTV) (SA-2 strain) genomic DNA (M. Johnson etal., 1994, Genbank sequence accession No. =L31965), prepared accordingto the technique of Example 2, and with the following oligonucleotides:

[0119] AB164 (33 mer) (SEQ ID No. 29)

[0120] 5′ CCGGTCGACATGGACCGCCATTTATTTTTGAGG 3′

[0121] AB165 (33 mer) (SEQ ID No. 30)

[0122] 5′ GGAAGATCTTTACGATGCTCCAAACCAGTAGCC 3′

[0123] so as to isolate the gene encoding the ILTV virus gD glycoproteinin the form of an SalI-BglII fragment. After purification, the 1134 bpPCR product was digested with SalI and BglII in order to isolate a 1122bp SalI-BglII fragment. This fragment was ligated with the vectorpVR1012 (Example 6), previously digested with SalI-BglII, to give theplasmid pAB089 (5984 bp) (FIG. No. 19).

EXAMPLE 21 Construction of the Plasmid pAB086 (AEV env Gene)

[0124] An RT-PCR reaction according to the technique of Example 5 wascarried out with the avian encephalomyelitis virus (AEV) (Type C)genomic RNA (E. Bieth et al., Nucleic Acids Res., 1992, 20, 367),prepared according to the technique of Example 3, and with the followingoligonucleotides:

[0125] AB160 (54 mer) (SEQ ID No. 31)

[0126] 5′ TTTGATATCATGGAAGCCGTCATTAAGGCATTTCTGACTGGATACCCTGGGAA

[0127] G3′

[0128] AB161 (31 mer) (SEQ ID No. 32)

[0129] 5′ TTTGGATCCTTATACTATTCTGCTTTCAGGC 3′

[0130] so as to isolate the sequence encoding the AEV virus Envglycoprotein in the form of an EcoRV-BamHI fragment. After purification,the 1836 bp RT-PCR product was digested with EcoRV and BamHI in order toisolate a 1825 bp EcoRV-BamHI fragment. This fragment was ligated withthe vector pVR1012 (Example 6), previously digested with EcoRV andBamHI, to give the plasmid pAB086 (6712 bp) (FIG. No. 20).

EXAMPLE 22 Construction of the Plasmid pAB081 (AEV gag/pro Gene)

[0131] An RT-PCR reaction according to the technique of Example 5 wascarried out with the avian encephalomyelitis virus (AEV) (Type C)genomic RNA (E. Bieth et al., Nucleic Acids Res., 1992, 20, 367),prepared according to the technique of Example 3, and with the followingoligonucleotides:

[0132] ABS150 (31 mer) (SEQ ID No. 33)

[0133] 5′ ACGCGTCGACATGGAAGCCGTCATTAAGGTG 3′

[0134] AB151 (32 mer) (SEQ ID No. 34)

[0135] 5′ TGCTCTAGACTATAAATTTGTCAAGCGGAGCC 3′

[0136] so as to isolate the sequence encoding the AEV virus Gag and Proproteins in the form of an SalI-XbaI fragment. After purification, the2125 bp RT-PCR product was digested with SalI-XbaI in order to isolate a2111 bp SalI-XbaI fragment. This fragment was ligated with the vectorpVR1012 (Example 6), previously digested with SalI and XbaI, to give theplasmid pAB081 (6996 bp) (FIG. No. 21).

EXAMPLE 23 Construction of the Plasmid pAB082 (Pneumovirus G Gene)

[0137] An RT-PCR reaction according to the technique of Example 5 wascarried out with the turkey rhinotracheitis virus (TRV) (2119 strain)genomic RNA (K. Juhasz et al., J. Gen. Virol., 1994, 75. 2873-2880),prepared according to the technique of Example 3, and with the followingoligonucleotides:

[0138] AB152 (32 mer) (SEQ ID No. 35)

[0139] 5′ AAACTGCAGAGATGGGGTCAGAGCTCTACATC 3′

[0140] AB153 (31 mer) (SEQ ID No. 36)

[0141] 5′ CGAAGATCTTTATTGACTAGTACAGCACCAC 3′

[0142] so as to isolate the gene encoding the TRV virus G glycoproteinin the form of a PstI-BglII fragment. After purification, the 2165 bpRT-PCR product was digested with PstI and BglII in order to isolate a1249 bp PstI-BglII fragment. This fragment was ligated with the vectorpVR1012 (Example 6), previously digested with PstI and BglII, to givethe plasmid pAB082 (6101 bp) (FIG. No. 22).

EXAMPLE 24 Construction of the Plasmid pAB077 (Avian Plague HA Gene,H2N2 Strain)

[0143] An RT-PCR reaction according to the technique of Example 5 wascarried out with the avian plague virus (AIV) (H2N2 Postdam strain)genomic RNA (J. Schäfer et al., Virology, 1993, 194, 781-788), preparedaccording to the technique of Example 3, and with the followingoligonucleotides:

[0144] AB142 (33 mer) (SEQ ID No. 37)

[0145] 5′ AAACTGCAGCAATGGCCATCATTTATCTAATTC 3′

[0146] AB143 (31 mer) (SEQ ID No. 38)

[0147] 5′ CGAAGATCTTCATATGCAGATTCTGCATTGC 3′

[0148] so as to isolate the gene encoding the HA glycoprotein from theavian plague virus (H2N2 strain) in the form of a PstI-BglII fragment.After purification, the 1709 bp RT-PCR product was digested with PstIand BglII in order to isolate a 1693 bp PstI-BglII fragment. Thisfragment was ligated with the vector pVR1012 (Example 6), previouslydigested with PstI and BglII, to give the plasmid pAB077 (6545 bp) (FIG.No. 23).

EXAMPLE 25 Construction of the Plasmid pAB078 (Avian Plague HA Gene,H7N7 Strain)

[0149] An RT-PCR reaction according to the technique of Example 5 wascarried out with the avian plague virus (AIV) (H7N7 Leipzig strain)genomic RNA (C. Rohm et al., Virology, 1995, 209, 664-670), preparedaccording to the technique of Example 3, and with the followingoligonucleotides:

[0150] AB144 (31 mer) (SEQ ID No. 39)

[0151] 5′ AAACTGCAGATGAACACTCAAATCCTGATAC 3′

[0152] AB145 (31 mer) (SEQ ID No. 40)

[0153] 5′ TTTGGATCCTTATATACAAATAGTGCACCGC 3′

[0154] so as to isolate the gene encoding the HA glycoprotein from theavian plague virus (H7N7 strain) in the form of a PstI-BamHI fragment.After purification, the 1707 bp RT-PCR product was digested with PstIand BamHI in order to isolate a 1691 bp PstI-BamHI fragment. Thisfragment was ligated with the vector pVR1012 (Example 6), previouslydigested with PstI and BamHI, to give the plasmid pAB078 (6549 bp) (FIG.No. 24).

EXAMPLE 26 Construction of the Plasmid pAB088 (Avian Plague NP Gene,H1N1 Strain)

[0155] An RT-PCR reaction according to the technique of Example 5 wascarried out with the avian influenza virus (AIV) (H1N1 Bavaria strain)genomic RNA (M. Gammelin et al., Virology, 1989, 170, 71-80), preparedaccording to the technique of Example 3, and with the followingoligonucleotides:

[0156] AB156 (32 mer) (SEQ ID No. 41)

[0157] 5′ CCGGTCGACATGGCGTCTCAAGGCACCAAACG 3′

[0158] AB158 (30 mer) (SEQ ID No. 42)

[0159] 5′ CGCGGATCCTTAATTGTCATACTCCTCTGC 3′

[0160] so as to isolate the gene encoding the avian influenza virus NPnucleoprotein in the form of a SalI-BamHI fragment. After purification,the 1515 bp RT-PCR product was digested with SalI and BamHI in order toisolate a 1503 bp SalI-BamHI fragment. This fragment was ligated withthe vector pVR1012 (Example 6), previously digested with SalI and BamHI,to give the plasmid pAB088 (6371 bp) (FIG. No. 25).

EXAMPLE 27 Construction of the Plasmid pAB079 (Avian Plague N Gene, H7N1Strain)

[0161] An RT-PCR reaction according to the technique of Example S wascarried out with the avian plague virus (AIV) (H7N1 Rostock strain)genomic RNA (J. McCauley, 1990, Genbank sequence accession No.=X52226),prepared according to the technique of Example 3, and with the followingoligonucleotides:

[0162] AB146 (35 mer) (SEQ ID No. 43)

[0163] 5′ CGCGTCGACATGAATCCAAATCAGAAAATAATAAC 3′

[0164] AB147 (31 mer) (SEQ ID No. 44)

[0165] 5′ GGAAGATCTCTACTTGTCAATGGTGAATGGC 3′

[0166] so as to isolate the gene encoding the N glycoprotein from theavian plague virus (H7Nl strain) in the form of an SalI-BglII fragment.After purification, the 1361 bp RT-PCR product was digested with SalIand BglII in order to isolate a 1350 bp SalI-BglII fragment. Thisfragment was Ligated with the vector pVR1012 (Example 6), previouslydigested with Sal1I and BglII, to give the plasmid pAB079 (6212 bp)(FIG. No. 26).

EXAMPLE 28 Preparation and Purification of the Plasmids

[0167] For the preparation of the plasmids intended for the vaccinationof animals, any technique may be used which makes it possible to obtaina suspension of purified plasmids predominantly in the supercoiled form.These techniques are well known to persons skilled in the art. There maybe mentioned in particular the alkaline lysis technique followed by twosuccessive ultracentrifugations on a caesium chloride gradient in thepresence of ethidium bromide as described in J. Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989). Reference may also be madeto patent applications PCT WO 95/21250 and PCT WO 96/02658 whichdescribe methods for producing, on an industrial scale, plasmids whichcan be used for vaccination. For the purposes of the manufacture ofvaccines (see Example 17), the purified plasmids are resuspended so asto obtain solutions at a high concentration (>2 mg/ml) which arecompatible with storage. To do this the plasmids are resuspended eitherin ultrapure water or in TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8.0).

EXAMPLE 29 Manufacture of the Associated Vaccines

[0168] The various plasmids necessary for the manufacture of anassociated vaccine are mixed starting with their concentrated solutions(Example 16). The mixtures are prepared such that the finalconcentration of each plasmid corresponds to the effective dose of eachplasmid. The solutions which can be used to adjust the finalconcentration of the vaccine may be either a 0.9% NaCl solution, or PBSbuffer.

[0169] Specific formulations such as liposomes, cationic lipids, mayalso be used for the manufacture of the vaccines.

EXAMPLE 30 Vaccination of Chickens

[0170] The chickens are vaccinated with doses of 10, 50 or 100 μg perplasmid. The injections can be performed with a needle by theintramuscular route. The sites of injection are the carina (for chickensmore than 2 weeks old) and the thigh (for 1-day-old or older chickens).In this case, the vaccinal doses are administered in the volume of 0.1to 0.3 ml.

[0171] In adult chickens (more than 20 weeks old) the injections arealso performed by the intramuscular route using a liquid jet injectionapparatus (with no needle) which has been specially designed for thevaccination of chickens (for example AVIJET apparatus). In this case,the injected volume is 0.3 ml. The injection may be performed in thecarina or at the level of the thigh. Likewise, in adult chickens, theinjections may be performed with a needle by the intramuscular route, inthe carina or in the thigh, in a volume of 0.3 ml. The injection of theplasmid vaccines can also be done in ovo. In this case, specialformulations as mentioned in Example 29 may be used. The volume injectedinto the 18-day embryonated egg is between 50 μl and 200 μl.

1 44 1 37 DNA Marek′s disease gammaherpesvirus MKT-1 1 aaaactgcagactatgcact attttaggcg gaattgc 37 2 35 DNA Marek′s diseasegammaherpesvirus MKT-1 2 ggaagatctt tacacagcat catcttctga gtctg 35 3 29DNA Marek′s disease gammaherpesvirus MKT-1 3 aaactgcaga tgaaagtatttttttttag 29 4 32 DNA Marek′s disease gammaherpesvirus MKT-1 4ggaagatctt tataggcggg aatatgcccg tc 32 5 39 DNA Newcastle disease virus5 ataagaatgc ggccgccatg gaccgtgcag ttagcagag 39 6 34 DNA Newcastledisease virus 6 cgcggatcct taaatcccat catccttgag aatc 34 7 1716 DNANewcastle disease virus 7 atggaccgtg cagttagcag agttgcgcta gagaatgaagaaagagaagc aaagaataca 60 tggcgctttg tattccggat tgcaatctta cttttaatagtaacaacctt agccatctct 120 gcaaccgccc tggtatatag catggaggct agcacgcctggcgaccttgt tggcataccg 180 actatgatct ctaaggcaga agaaaagatt acatctgcactcagttctaa tcaagatgta 240 gtagatagga tatataagca ggtggccctt gagtctccattggcgttgct aaacactgaa 300 tctgtaatta tgaatgcaat aacgtctctc tcttatcaaatcaatggagc tgcaaataat 360 agcgggtgtg gggcacctgt tcatgaccca gattatatcggggggatagg caaagaactt 420 attgtggatg acgctagtga tgtcacatca ttctatccctctgcgttcca agaacacctg 480 aactttatcc cggcacctac tacaggatca ggttgcactcggataccctc attcgacata 540 agcgctaccc actactgtta cactcacaat gtgatattatctggttgcag agatcactca 600 cactcatatc agtacttagc acttggcgtg cttcggacatctgcaacagg gagggtattc 660 ttttctactc tgcgttccat caatttggat gacagccaaaatcggaagtc ttgcagtgtg 720 agtgcaactc ccttaggttg tgatatgctg tgctctaaaatcacagagac tgaggaagag 780 gattatagtt caattacgcc tacatcgatg gtgcacggaaggttagggtt tgacggtcaa 840 taccatgaga aggacttaga cgtcataact ttatttaaggattgggtggc aaattaccca 900 ggagtggggg gtgggtcttt tattaacaac cgcgtatggttcccagtcta cggagggcta 960 aaacccaatt cgcctagtga caccgcacaa gaagggagatatgtaatata caagcgctac 1020 aatgacacat gcccagatga acaagattac cagattcggatggctaagtc ttcatataag 1080 cctgggcggt ttggtggaaa acgcgtacag caggccatcttatctatcaa ggtgtcaaca 1140 tctttgggcg aggacccggt gctgactgta ccgcctaatacaatcacact catgggggcc 1200 gaacggagag ttctcacagt agggacatct catttcttgtaccagcgagg gtcttcatac 1260 ttctctcctg ctttattata ccctatgaca gtcaacaacaaaacggctac tcttcatagt 1320 ccttacacat tcaatgcttt cactaggcca ggtagtgtcccttgtcaggc atcagcaaga 1380 tgccccaact catgtgtcac tggagtttat actgatccgtatcccttagt cttccatagg 1440 aaccatacct tgcggggggt attcgggaca atgcttgatgatgaacaagc aagacttaac 1500 cctgtatctg cagtatttga taacatatcc cgcagtcgcataacccgggt aagttcaagc 1560 cgtactaagg cagcatacac gacatcgaca tgttttaaagttgtcaagac caataaaaca 1620 tattgcctca gcattgcaga aatatccaat accctcttcggggaattcag gatcgttcct 1680 ttactagttg agattctcaa ggatgatggg atttaa 17168 37 DNA Newcastle disease virus 8 agaatgcggc cgcgatgggc tccagatcttctaccag 37 9 34 DNA Newcastle disease virus 9 tgctctagat catatttttgtagtggctct catc 34 10 1662 DNA Newcastle disease virus 10 atgggctccagatcttctac caggatcccg gtacctctaa tgctgatcat ccgaaccgcg 60 ctgacactgagctgtatccg tctgacaagc tctcttgatg gcaggcctct tgcggctgca 120 gggatcgtggtaacaggaga taaagcagtc aacatataca cctcatccca gacagggtca 180 atcatagttaagttactccc gaatatgccc aaggacaaag aggtgtgtgc aaaagcccca 240 ttggaggcatacaacaggac actgactact ttactcaccc cccttggtga ttctatccgc 300 aggatacaagagtctgtgac tacttccgga ggaaggagac agagacgctt tataggtgcc 360 attatcggcagtgtagctct tggggttgcg acagctgcac agataacagc agcttcggcc 420 ctgatacaagccaaccagaa tgctgccaac atcctccggc ttaaagagag cattgctgca 480 accaatgaagctgtgcacga ggtcactgac ggattatcac aactagcagt ggcagtaggg 540 aagatgcaacagtttgtcaa tgaccagttc aataatacag cgcaagaatt ggactgtata 600 aaaattgcacagcaggtcgg tgtagaactc aacttgtacc taactgaatt gactacagta 660 tttgggccacaaatcacttc ccctgcctta actcagctga ctatccaagc gctttacaat 720 ctagctggtggtaatatgga ttacttgctg actaagttag gtgtagggaa caaccaactc 780 agctcattaattggtagcgg cttgatcacc ggcaacccta ttctgtacga ctcacagact 840 cagatcttgggtatacaggt aactttgcct tcagttggga acctgaataa tatgcgtgcc 900 acctacctggagaccttatc tgtaagcaca accaagggat ttgcctcagc acttgtccca 960 aaagtggtgacacaggtcgg ttccgtgata gaagaacttg acacctcata ctgtataggg 1020 accgacttggatttatactg tacaagaata gtgacattcc ctatgtctcc tggtatttat 1080 tcttgtctgagcggtaatac atcggcttgc atgtattcaa agactgaagg cgcacttact 1140 acgccatatatggctctcaa aggctcagtt attgccaatt gcaagctgac aacatgtaga 1200 tgtgcagatcccccaggtat catatcgcaa aattatggag aagctgtgtc cttaatagat 1260 aggcactcatgcaacgtctt atccttagac gggataactc tgaggctcag tggggaattt 1320 gatgcaacctatcaaaagaa tatctctata ctagattctc aagttatagt gacaggcaat 1380 cttgatatatcaactgagct tgggaatgtc aacaactcaa taagtaatgc cctgaataag 1440 ttagaggaaagcaacagcaa actagacaaa gtcaatgtca aactgaccag cacatctgct 1500 ctcattacctacatcgtttt aactgtcata tctcttgttt ttggtgtact tagcctggtt 1560 ctagcatgctacctgatgta caagcaaaag gcacaacaaa agaccttgtt atggcttggg 1620 aataatacccttgatcagat gagagccact acaaaaatat ga 1662 11 33 DNA Infectious bursaldisease virus 11 tcagatatcg atgacaaacc tgcaagatca aac 33 12 38 DNAInfectious bursal disease virus 12 agaatgcggc cgcttacctc cttatagcccggattatg 38 13 1362 DNA Infectious bursal disease virus 13 atgacaaacctgcaagatca aacccaacag attgttccgt tcatacggag ccttctgatg 60 ccaacaaccggaccggcgtc cattccggac gacaccctgg agaagcacac tctcaggtca 120 gagacctcgacctacaattt gactgtgggg gacacagggt cagggctaat tgtctttttc 180 cctggattccctggctcaat tgtgggtgct cactacacac tgcagagcaa tgggaactac 240 aagttcgatcagatgctcct gactgcccag aacctaccgg ccagctacaa ctactgcaga 300 ctagtgagtcggagtctcac agtgaggtca agcacactcc ctggtggcgt ttatgcacta 360 aacggcaccataaacgccgt gaccttccaa ggaagcctga gtgaactgac agatgttagc 420 tacaatgggttgatgtctgc aacagccaac atcaacgaca aaattgggaa tgtcctggta 480 ggggaaggggtcactgtcct cagcctaccc acatcatatg atcttgggta tgtgaggctt 540 ggtgaccccattcccgctat agggcttgac ccaaaaatgg tagctacatg cgacagcagt 600 gacaggcccagagtctacac cataactgca gccgatgatt accaattctc atcacagtac 660 caaccaggtggggtaacaat cacactgttc tcagccaaca ttgatgctat cacaagcctc 720 agcattgggggagagctcgt gtttcaaaca agcgtccaag gccttgtact gggcgccacc 780 atctaccttataggctttga tgggactgcg gtaatcacca gagctgtagc cgcagataat 840 gggctgacggccggcaccga caatcttatg ccattcaatc ttgtcattcc aaccaatgag 900 ataacccagccaatcacatc catcaaactg gagatagtga cctccaaaag tggtggtcag 960 gcaggggatcagatgtcatg gtcggcaagt gggagcctag cagtgacgat ccatggtggc 1020 aactatccaggggccctccg tcccgtcaca ctagtagcct acgaaagagt ggcaacagga 1080 tccgtcgttacggtcgctgg ggtgagtaac ttcgagctga ttccaaatcc tgaactagca 1140 aagaacctggttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg 1200 atactgagtgagagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact 1260 gattttcgtgagtacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga 1320 gcatttggcttcaaagacat aatccgggct ataaggaggt aa 1362 14 32 DNA chicken infectiousbronchitis virus 14 acgcgtcgac atgttggtaa cacctctttt ac 32 15 35 DNAchicken infectious bronchitis virus 15 ggaagatctt cattaacgtc taaaacgacgtgttc 35 16 1614 DNA chicken infectious bronchitis virus 16 atgttggtaacacctctttt actagtgact cttttgtgtg tactatgtag tgctgctttg 60 tatgacagtagttcttacgt ttactactac caaagtgcct ttagaccacc taatggttgg 120 catttacacgggggtgctta tgcggtagtt aatatttcta gcgaatctaa taatgcaggc 180 tcttcacctgggtgtattgt tggtactatt catggtggtc gtgttgttaa tgcttcttct 240 atagctatgacggcaccgtc atcaggtatg gcttggtcta gcagtcagtt ttgtactgca 300 cactgtaacttttcagatac tacagtgttt gttacacatt gttataaata tgatgggtgt 360 cctataactggcatgcttca aaagaatttt ttacgtgttt ctgctatgaa aaatggccag 420 cttttctataatttaacagt tagtgtagct aagtacccta cttttaaatc atttcagtgt 480 gttaataatttaacatccgt atatttaaat ggtgatcttg tttacacctc taatgagacc 540 acagatgttacatctgcagg tgtttatttt aaagctggtg gacctataac ttataaagtt 600 atgagagaagttaaagccct ggcttatttt gttaatggta ctgcacaaga tgttattttg 660 tgtgatggatcacctagagg cttgttagca tgccagtata atactggcaa tttttcagat 720 ggcttttatccttttattaa tagtagttta gttaagcaga agtttattgt ctatcgtgaa 780 aatagtgttaatactacttt tacgttacac aatttcactt ttcataatga gactggcgcc 840 aaccctaatcctagtggtgt tcagaatatt ctaacttacc aaacacaaac agctcagagt 900 ggttattataattttaattt ttcctttctg agtagttttg tttataagga gtctaatttt 960 atgtatggatcttatcaccc aagttgtaat tttagactag aaactattaa taatggcttg 1020 tggtttaattcactttcagt ttcaattgct tacggtcctc ttcaaggtgg ttgcaagcaa 1080 tctgtctttagtggtagagc aacttgttgt tatgcttatt catatggagg tccttcgctg 1140 tgtaaaggtgtttattcagg tgagttagct cttaattttg aatgtggact gttagtttat 1200 gttactaagagcggtggctc tcgtatacaa acagccactg aaccgccagt tataactcga 1260 cacaattataataatattac tttaaatact tgtgttgatt ataatatata tggcagaact 1320 ggccaaggttttattactaa tgtaaccgac tcagctgtta gttataatta tctagcagac 1380 gcaggtttggctattttaga tacatctggt tccatagaca tctttgttgt acaaggtgaa 1440 tatggtcttacttattataa ggttaaccct tgcgaagatg tcaaccagca gtttgtagtt 1500 tctggtggtaaattagtagg tattcttact tcacgtaatg agactggttc tcagcttctt 1560 gagaaccagttttacattaa aatcactaat ggaacacgtc gttttagacg ttaa 1614 17 39 DNA chickeninfectious bronchitis virus 17 ataagaatgc ggccgcatgt ccaacgagacaaattgtac 39 18 38 DNA chicken infectious bronchitis virus 18 ataagaatgcggccgcttta ggtgtaaaga ctactccc 38 19 678 DNA chicken infectiousbronchitis virus 19 atgtccaacg agacaaattg tactcttgac tttgaacagtcagttgagct ttttaaagag 60 tataatttat ttataactgc attcttgttg ttcttaaccataatacttca gtatggctat 120 gcaacaagaa gtaagtttat ttatatactg aaaatgatagtgttatggtg cttttggccc 180 cttaacattg cagtaggtgt aatttcatgt atatacccaccaaacacagg aggtcttgtc 240 gcagcgataa tacttacagt gtttgcgtgt ctgtcttttgtaggttattg gatccagagt 300 attagactct ttaagcggtg taggtcatgg tggtcatttaacccagaatc taatgccgta 360 ggttcaatac tcctaactaa tggtcaacaa tgtaattttgctatagagag tgtgccaatg 420 gtgctttctc caattataaa gaatggtgtt ctttattgtgagggtcagtg gcttgctaag 480 tgtgaaccag accacttgcc taaagatata tttgtttgtacaccggatag acgtaatatc 540 taccgtatgg tgcagaaata tactggtgac caaagcggaaataagaaacg gtttgctacg 600 tttgtctatg caaagcagtc agtagatact ggcgagctagaaagtgtagc aacaggaggg 660 agtagtcttt acacctaa 678 20 34 DNA chickeninfectious bronchitis virus 20 aaaactgcag tcatggcaag cggtaaggca actg 3421 33 DNA chicken infectious bronchitis virus 21 cgcggatcct caaagttcattctctcctag ggc 33 22 1230 DNA chicken infectious bronchitis virus 22atggcaagcg gtaaggcaac tggaaagaca gacgccccag ctccagtcat caaactagga 60ggaccaaagc cacctaaagt tggttcttct ggaaatgtat cttggtttca agcaataaaa 120gccaagaagt taaattcacc tccgcctaag tttgaaggta gcggtgttcc tgataatgaa 180aatctaaaac caagtcagca gcatggatat tggagacgcc aagctaggtt taagccaggt 240aaaggtggaa gaaaaccagt cccagatgct tggtattttt actatactgg aacaggacca 300gccgctaacc tgaattgggg tgatagccaa gatggtatag tgtgggttgc tggtaagggt 360gctgatacta aatttagatc taatcagggt actcgtgact ctgacaagtt tgaccaatat 420ccgctacggt tttcagacgg aggacctgat ggtaatttcc gttgggattt cattcctctg 480aatcgtggca ggagtgggag atcaacagca gcttcatcag cggcatctag tagagcacca 540tcacgtgaag tttcgcgtgg tcgcaggagt ggttctgaag atgatcttat tgctcgtgca 600gcaaggataa ttcaggatca gcagaagaag ggttctcgca ttacaaaggc taaggctgat 660gaaatggctc accgccggta ttgcaagcgc actattccac ctaattataa ggttgatcaa 720gtgtttggtc cccgtactaa aggtaaggag ggaaattttg gtgatgacaa gatgaatgag 780gaaggtatta aggatgggcg cgttacagca atgctcaacc tagttcctag cagccatgct 840tgtcttttcg gaagtagagt gacgcccaga cttcaaccag atgggctgca cttgaaattt 900gaatttacta ctgtggtccc acgtgatgat ccgcagtttg ataattatgt aaaaatttgt 960gatcagtgtg ttgatggtgt aggaacacgt ccaacagatg atgaaccaag accaaagtca 1020cgctcaagtt caaaacctgc aacaagagga aattctccag cgccaagaca gcagcgccct 1080aagaaggaga aaaagccaaa gaagcaggat gatgaagtgg ataaagcatt gacctcagat 1140gaggagagga acaatgcaca gctggaattt gatgatgaac ccaaggtaat taactggggg 1200gattcagccc taggagagaa tgaactttga 1230 23 39 DNA Chicken anemia virus 23ttcttgcggc cgccatggca agacgagctc gcagaccga 39 24 38 DNA Chicken anemiavirus 24 ttcttgcggc cgctcagggc tgcgtccccc agtacatg 38 25 39 DNA Chickenanemia virus 25 ttcttgcggc cgccatgcac gggaacggcg gacaaccgg 39 26 32 DNAChicken anemia virus 26 cgcggatcct cacactatac gtaccggggc gg 32 27 38 DNAchicken infectious laryngotracheitis virus 27 ttcttgcggc cgccatggctagcttgaaaa tgctgatc 38 28 36 DNA chicken infectious laryngotracheitisvirus 28 ttcttgcggc cgcttattcg tcttcgcttt cttctg 36 29 33 DNA chickeninfectious laryngotracheitis virus 29 ccggtcgaca tggaccgcca tttatttttgagg 33 30 33 DNA chicken infectious laryngotracheitis virus 30ggaagatctt tacgatgctc caaaccagta gcc 33 31 54 DNA avianencephalomyelitis virus 31 tttgatatca tggaagccgt cattaaggca tttctgactggataccctgg gaag 54 32 31 DNA avian encephalomyelitis virus 32 tttggatccttatactattc tgctttcagg c 31 33 31 DNA avian encephalomyelitis virus 33acgcgtcgac atggaagccg tcattaaggt g 31 34 32 DNA avian encephalomyelitisvirus 34 tgctctagac tataaatttg tcaagcggag cc 32 35 32 DNA Turkeyrhinotracheitis virus 35 aaactgcaga gatggggtca gagctctaca tc 32 36 31DNA Turkey rhinotracheitis virus 36 cgaagatctt tattgactag tacagcacca c31 37 33 DNA avian plague virus 37 aaactgcagc aatggccatc atttatctaa ttc33 38 31 DNA avian plague virus 38 cgaagatctt catatgcaga ttctgcattg c 3139 31 DNA avian plague virus 39 aaactgcaga tgaacactca aatcctgata c 31 4031 DNA avian plague virus 40 tttggatcct tatatacaaa tagtgcaccg c 31 41 32DNA Avian influenza virus 41 ccggtcgaca tggcgtctca aggcaccaaa cg 32 4230 DNA Avian influenza virus 42 cgcggatcct taattgtcat actcctctgc 30 4335 DNA avian plague virus 43 cgcgtcgaca tgaatccaaa tcagaaaata ataac 3544 31 DNA avian plague virus 44 ggaagatctc tacttgtcaa tggtgaatgg c 31

1. Avian vaccine formula, comprising at least three polynucleotidevaccine valencies each comprising a plasmid integrating, so as toexpress it in viva in the host cells, a gene with one avian pathogenvalency, these valencies being selected from the group consisting ofMarek's disease virus, Newcastle disease virus, infectious bursaldisease virus, infectious anaemia virus, the plasmids comprising, foreach valency, one or more of the genes selected from the groupconsisting of gB and gD for the Marek's disease virus, HN and F for theNewcastle disease virus, VP2 for the infectious bursal disease virus,C+NS1 for the infectious anaemia virus.
 2. Vaccine formula according toclaim 1, wherein, for the valency of the Marek's disease virus, itcomprises the gB gene alone.
 3. Formula according to claim 1, whichcomprises the Newcastle disease virus HN and F genes in the same plasmidor in different plasmids.
 4. Vaccine formula according to claim 1,wherein the plasmid for the infectious anaemia virus comprises C+NS1 inthe same plasmid.
 5. Vaccine formula according to any one of claims 1 to4, which comprises, in addition, at least one valency selected from thegroup consisting of infectious bronchitis virus, infectiouslaryngotracheitis virus, encephalomyelitis virus, pneumovirosis virus,and avian plague virus, the plasmids comprising, for these valencies,one or more of the genes selected from the group consisting of S, M andN for the infectious bronchitis virus, gB and gD for the infectiouslaryngotracheitis virus, env and gag/pro for the encephalomyelitisvirus, F and G for the pneumovirosis virus and HA, N and NP for theavian plague virus.
 6. Vaccine formula according to claim 5, wherein forthe infectious bronchitis virus valency, it comprises the S gene alone.7. Vaccine formula according to claim 5, wherein for thelaryngotracheitis valency, the formula comprises the gB gene alone. 8.Vaccine formula according to claim 5, wherein for the pneumovirosisvalency, the formula comprises the two F and G genes in differentplasmids or in one and the same plasmid.
 9. Vaccine formula according toclaim 5, wherein, for the avian plague valency, the formula comprisesthe HA gene alone.
 10. Vaccine formula according to claim 5, wherein forthe encephalomyelitis valency, the vaccine formula comprises the envgene.
 11. Vaccine formula according to any one of claims 1 to 10, whichcomprises from 10 ng to 1 mg, preferably from 100 ng to 500 μg, stillmore preferably from 0.1 μg to 50 μg of each plasmid.
 12. Use of one ormore plasmids as described in any one of claims 1 to 11, for themanufacture of an avian vaccine intended to vaccinate animals firstvaccinated by means of a first vaccine selected from the groupconsisting of a live whole vaccine, an inactivated whole vaccine, asubunit vaccine, a recombinant vaccine, this first vaccine having theantigen(s) encoded by the plasmid(s) or antigen(s) providingcross-protection.
 13. Vaccination kit grouping together a vaccineformula according to any one of claims 1 to 11, and an avian vaccineselected from the group consisting of a live whole vaccine, aninactivated whole vaccine, a subunit vaccine, a recombinant vaccine,this first vaccine having the antigen encoded by the polynucleotidevaccine or an antigen providing cross-protection, for an administrationof the latter in first vaccination or as booster with the vaccineformula.
 14. Vaccine formula according to any one of claims 1 to 11,accompanied by a leaflet indicating that this formula can be used asbooster for a first avian vaccine selected from the group consisting ofa live whole vaccine, an inactivated whole vaccine, a subunit vaccine, arecombinant vaccine, this first vaccine having the antigen encoded bythe polynucleotide vaccine or an antigen providing cross-protection.